Ribosomal RNA (rRNA) sequences are present in all prokaryotic and eukaryotic cells. These sequences are highly conserved among organisms and are know to be species specific. Sequence analysis is widely utilized in diagnostic microbiology in order to identify infectious agents. A rapid in situ hybridization assay for detection of rRNA sequences in formalin or Bouin's fixed paraffin embedded tissues was developed using biotin-labeled oligonucleotide DNA probes specific for a variety of rRNA sequences. This method was used effectively to identify human, Aspergillus species, Pneumocystis carinii, and Toxoplasma gondii rRNA sequences in a protocol requiring less than 30min to complete. Utilization of site specific oligonucleotide probes specific for a variety of rRNA sequences may aid in the diagnosis of several medically important bacterial, fungal, and protozoal pathogens.